Liposomes with lipids having advantageous pka-value for RNA delivery

Abstract

To provide a composition containing liposomes and RNA molecules encoding an immunogen.SOLUTION: RNA encoding an immunogen is delivered in a liposome for the purposes of immunisation. The liposome includes lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These liposomes can have essentially neutral surface charge at physiological pH and are effective for immunisation. In one embodiment, a liposome having a lipid bilayer encapsulating an aqueous core is provided, wherein: (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6; and (ii) the aqueous core includes a RNA which encodes an immunogen.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 61 / 361,830, filed July 6, 2010, and U.S. Provisional Application No. 61 / 378,837, filed August 31, 2010, both of which are incorporated herein by reference in their entirety for all purposes.

[0002] (Technical field) The present invention is in the field of non-viral delivery of RNA for immunization. [Background technology]

[0003] (Background technology) Delivery of nucleic acids to immunize animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, viral or non-viral delivery vehicles (or even no delivery vehicle, in a "naked" vaccine), replicating or non-replicating vectors, or viral or non-viral vectors.

[0004] There remains a need for further improved nucleic acid vaccines. Summary of the Invention [Means for solving the problem]

[0005] DISCLOSURE OF THEINVENTION According to the present invention, RNA encoding an immunogen is delivered in a liposome for immunization purposes. The liposome comprises a lipid having a pKa in the range of 5.0-7.6. Ideally, lipids with a pKa in this range have a tertiary amine; such lipids behave differently than lipids with quaternary amine groups (e.g., DOTAP or DC-Chol). At physiological pH, amines with a pKa in the range of 5.0-7.6 have a neutral or reduced surface charge, whereas lipids such as DOTAP are strongly cationic. The present inventors have found that liposomes formed from quaternary amine lipids (e.g., DOTAP) are less suitable for delivery of RNA encoding an immunogen than liposomes formed from tertiary amine lipids (e.g., DLinDMA).

[0006] Thus, the present invention provides liposomes having a lipid bilayer encapsulating an aqueous core, where: (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6, and preferably a lipid having a tertiary amine; and (ii) the aqueous core comprises RNA encoding an immunogen. Because these liposomes are suitable for in vivo delivery of the RNA to vertebrate cells, they are useful as components in pharmaceutical compositions for immunizing subjects against various diseases.

[0007] The invention also provides a process for preparing RNA-containing liposomes comprising: (a) mixing RNA with a lipid at a pH below the pKa of the lipid but above 4.5 to form a liposome in which the RNA is encapsulated; and (b) raising the pH of the resulting liposome-containing mixture above the pKa of the lipid.

[0008] (Liposomes) The present invention utilizes liposomes in which RNA encoding an immunogen is encapsulated. Thus, the RNA is separated from any external medium by the lipid bilayer of the liposome (as in natural viruses), and encapsulation in this manner has been found to protect the RNA from RNase digestion. Although the liposomes may contain some external RNA (e.g., on their surface), at least half of the RNA (and ideally, all of it) is encapsulated in the core of the liposome. Encapsulation within liposomes is different from lipid / RNA complexes, for example, as disclosed in Reference 1.

[0009] A variety of amphipathic lipids can form bilayers in an aqueous environment to encapsulate the RNA-containing aqueous core as a liposome. These lipids can have anionic, cationic or zwitterionic hydrophilic head groups. The liposomes of the present invention include lipids with a pKa in the range of 5.0 to 7.6, with preferred lipids with a pKa in this range having a tertiary amine. For example, they can include 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA; pKa 5.8) and / or 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Another suitable lipid with a tertiary amine is 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA). See FIG. 3 and reference 2. Some of the amino acid lipids of reference 3 can also be used, as can certain of the amino lipids of reference 4. Further useful lipids having tertiary amines in their headgroups are disclosed in reference 5, the entire contents of which are incorporated herein by reference.

[0010] Liposomes of the invention can be formed from a single lipid or a mixture of lipids, provided that at least one of the lipids has a pKa (and preferably a tertiary amine) in the range of 5.0 to 7.6. Within this pKa range, preferred lipids have a pKa of 5.5 to 6.7 (e.g., between 5.6 to 6.8, between 5.6 to 6.3, between 5.6 to 6.0, between 5.5 to 6.2, or between 5.7 to 5.9). The pKa is the pH at which 50% of the lipids are charged, which is midway between the point at which the lipid is fully charged and the point at which the lipid is fully uncharged. The pKa is measured using the methods disclosed below in the section entitled "pKa Measurements." The pKa should typically be measured for the lipid alone, rather than for the lipid in the context of a mixture that also includes other lipids (e.g., not as is done in Reference 6, but rather as seen in the pKa of SNALPs rather than the pKa of individual lipids).

[0011] When the liposomes of the present invention are formed from a mixture of lipids, it is preferred that the percentage of those lipids having a pKa in the desired range should be between 20-80% (e.g., between 30-70%, or between 40-60%) of the total amount of lipid. For example, useful liposomes are shown below, in which 40% or 60% of the total lipids are lipids having a pKa in the desired range. The remainder can be made, for example, from cholesterol (e.g., 35-50% cholesterol) and / or DMG (optionally PEGylated) and / or DSPC. Such mixtures are used below. These percentages are molar percentages.

[0012] Liposomes may contain amphiphilic lipids whose hydrophilic portion is PEGylated (i.e., modified by the covalent attachment of polyethylene glycol). This modification may increase the stability of the liposomes and prevent nonspecific adsorption. For example, lipids may be conjugated to PEG using techniques such as those disclosed in references 6 and 7. PEG provides a coat that may impart advantageous pharmacokinetic properties to the liposomes. The combination of efficient encapsulation of RNA (especially self-replicating RNA), cationic lipids with pKa in the range of 5.0-7.6, and a PEGylated surface allows for efficient delivery to multiple cell types (including both immune and non-immune cells), thereby eliciting a stronger and better immune response than when using quaternary amines without PEGylation. PEGs of various lengths may be used, for example, between 0.5 and 8 kDa.

[0013] The lipids used in the present invention can be saturated or unsaturated.It is preferred to use at least one unsaturated lipid to prepare liposome.Figure 3 shows three useful unsaturated lipids.When an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail.

[0014] A mixture of DSPC, DLinDMA, PEG-DMG and cholesterol is used in the examples. An independent aspect of the invention is a liposome comprising DSPC, DLinDMA, PEG-DMG and cholesterol. The liposome preferably encapsulates RNA, such as a self-replicating RNA, e.g., encoding an immunogen.

[0015] Liposomal particles are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle, forming several distinct aqueous compartments. SUVs and LUVs have a single bilayer that encapsulates an aqueous core; SUVs typically have a diameter ≦50 nm, and LUVs have a diameter >50 nm. The liposomal particles of the present invention are ideally LUVs with a diameter in the range of 50-220 nm. For a composition comprising a population of LUVs with different diameters: (i) at least 80% (by number) should have a diameter in the range of 20-220 nm, (ii) the mean diameter (Zav (by intensity)) of said population is ideally in the range of 40-200 nm, and / or (iii) said diameters should have a polydispersity index <0.2. The liposome / RNA complexes of Reference 1 are predicted to have diameters in the range of 600-800 nm and to be highly polydisperse. The liposomes may be substantially spherical.

[0016] Techniques for preparing suitable liposomes are well known in the art (see, e.g., References 8-10). One useful method is described in Reference 11 and involves mixing (i) an ethanolic solution of lipids, (ii) an aqueous solution of nucleic acid, and (iii) a buffer solution, followed by mixing, equilibration, dilution, and purification steps. The preferred liposomes of the present invention can be obtained by this mixing process.

[0017] (Mixing process) As described above, the present invention provides a process for preparing RNA-containing liposomes comprising: (a) mixing RNA with a lipid at a pH below the pKa of the lipid but above 4.5; and then (b) raising the pH above the pKa of the lipid.

[0018] Thus, the cationic lipids become positively charged during liposome formation in step (a), but the subsequent pH change means that most (or all) of the positively charged groups become neutral. This process is advantageous for preparing liposomes of the invention, and by avoiding a pH below 4.5 during step (a), the stability of the encapsulated RNA is improved.

[0019] The pH in step (a) is above 4.5, ideally above 4.8. A pH in the range of 5.0 to 6.0, or in the range of 5.0 to 5.5 may be used to provide suitable liposomes.

[0020] The increased pH in step (b) is above the pKa of the lipid. The pH is ideally increased to a pH below 9, and preferably below 8. Depending on the pKa of the lipid, the pH in step (b) may therefore be increased to be within the range of 6 to 8, for example to pH 6.5±0.3. The pH increase in step (b) may be achieved by transferring the liposomes into a suitable buffer, for example to phosphate buffered saline. The pH increase in step (b) is ideally carried out after liposome formation has occurred.

[0021] The RNA used in step (a) may be in aqueous solution during mixing with an organic solution of the lipids (e.g., an ethanolic solution, as in reference 11). The mixture may then be diluted to form liposomes, after which the pH may be raised in step (b).

[0022] (RNA) The present invention is useful for in vivo delivery of RNA encoding an immunogen. The RNA is translated by non-immune cells at the delivery site, resulting in expression of the immunogen, and it also causes immune cells to secrete type I interferons and / or pro-inflammatory cytokines, which provide a local adjuvant effect. The non-immune cells can also secrete type I interferons and / or pro-inflammatory cytokines in response to the RNA.

[0023] Because the RNA is positive stranded, it can be translated by non-immune cells without the need for any intervening replication step (e.g., reverse transcription). It can also bind to the TLR7 receptor expressed by immune cells, thereby initiating an adjuvant effect.

[0024] Preferred positive strand RNAs are self-replicating. Even when self-replicating RNA molecules (replicons) are delivered to vertebrate cells without any proteins, they can result in the generation of multiple daughter RNAs by transcription from the self-replicating RNA molecule itself (through antisense copies generated from the self-replicating RNA molecule). Self-replicating RNA molecules are thus typically positive strand molecules that can be directly translated after delivery to a cell, providing an RNA-dependent RNA polymerase that then generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the generation of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded immunogen, or can be transcribed to provide additional transcripts of the same sense as the delivered RNA, which are translated to provide in situ expression of the immunogen. The overall result of this series of transcriptions is a vast amplification in the number of the introduced replicon RNAs, such that the encoded immunogen becomes the major polypeptide product of the cell.

[0025] As shown below, self-replicating activity is not required for RNA to provide an adjuvant effect, but it can enhance post-transfection secretion of cytokines. The self-replicating activity is particularly useful for achieving high-level expression of immunogens by non-immune cells. It can also enhance apoptosis of the non-immune cells.

[0026] One suitable system to achieve self-replication is to use alphavirus-based RNA replication. These positive-strand replicons are translated after delivery to the cell to give a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that self-cleaves to give a replication complex that creates a genomic negative-strand copy of the delivered positive-strand RNA. These negative-strand transcripts can themselves be transcribed to give further copies of the positive-strand parent RNA and also to give subgenomic transcripts that code for immunogens. Translation of the subgenomic transcripts thus results in in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons may use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences may be used (e.g., the attenuated TC83 mutant of VEEV has been used in replicons

[12] ).

[0027] A preferred self-replicating RNA molecule thus encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an immunogen. The polymerase may be an alphavirus replicase, such as one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0028] Whereas naturally occurring alphavirus genomes encode structural virion proteins in addition to the nonstructural replicase polyproteins, the self-replicating RNA molecules of the invention preferably do not encode alphavirus structural proteins. Thus, the preferred self-replicating RNA may result in the production of genomic RNA copies of itself in a cell, but not in the production of RNA-containing virions. The inability to generate these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules are unable to perpetuate themselves in an infectious form. The alphavirus structural proteins necessary for perpetuation in wild-type viruses are absent in the self-replicating RNA of the invention, and their location is dictated by the gene encoding the immunogen of interest. As a result, the subgenomic transcript encodes the immunogen, rather than the structural alphavirus virion proteins.

[0029] Thus, a self-replicating RNA molecule useful in the invention may have two open reading frames: the first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an immunogen. In some embodiments, the RNA may have an additional (e.g., downstream) open reading frame, e.g., to encode an additional immunogen (see below) or to encode an auxiliary polypeptide.

[0030] The self-replicating RNA molecule may have a 5' sequence that is compatible with the encoded replicase.

[0031] Self-replicating RNA molecules can have various lengths, but they are typically 5000 to 25000 nucleotides long (e.g., 8000 to 15000 nucleotides, or 9000 to 12000 nucleotides). Thus, the RNA is longer than that observed in siRNA delivery.

[0032] RNA molecules useful in the present invention can have a 5' cap (e.g., 7-methylguanosine), which can enhance in vivo translation of the RNA.

[0033] The 5' nucleotide of the RNA molecule useful in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to 7-methylguanosine via a 5' to 5' bridge. The 5' triphosphate may enhance RIG-I binding and thus promote the adjuvant effect.

[0034] The RNA molecule may have a 3' poly A tail. It may also contain a poly A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

[0035] The RNA molecules useful in the present invention are typically single-stranded. Single-stranded RNA can generally initiate adjuvant effects by binding to TLR7, TLR8, RNA helicase and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA formed either during replication of single-stranded RNA or within the secondary structure of single-stranded RNA.

[0036] RNA molecules useful in the present invention can be conveniently prepared by in vitro transcription (IVT). IVT can use (cDNA) templates made and propagated in bacteria in plasmid form or made synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering). For example, DNA-dependent RNA polymerases (e.g., bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe RNA from DNA templates. Appropriate capping and polyA addition reactions can be used if necessary (although the polyA of the replicon is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the 5' nucleotides transcribed, and in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT-transcribed RNA can function efficiently as a substrate for the self-encoded replicase.

[0037] As discussed in Reference 13, the self-replicating RNA may contain one or more nucleotides with modified nucleobases (in addition to any 5' cap structure). Thus, the RNA may contain: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (l-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine); ms2i6A (2-methylthio-N6 isopentenyl adenosine); io 6A(N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine);g6A(N6-glycinylcarbamoyladenosine);t6A(N6-threonylcarbamoyladenosine);ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine);m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6.-hydroxynorvalylcarbamoyladenosine);ms 2hn6A(2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine);Ar(p)(2'-O-ribosyladenosine(phosphate));I(inosine);m11(1-methylinosine);m'Im(1,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-O-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-formyl(fonnyl)cytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm (N4 acetyl-2TOmethylcytidine);k2C (lysidine);m1G (1-methylguanosine);m2G (N2-methylguanosine);m7G (7-methylguanosine);Gm (2'-O-methylguanosine);m22G (N2,N2-dimethylguanosine);m2Gm (N2,2'-O-dimethylguanosine);m22Gm (N2,N2,2'-O-trimethylguanosine);Gr(p) (2'-O-ribosylguanosine (phosphate));yW (wybutosine);o2yW (peroxywybutosine);OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine); m 5Um(5,2'-O-dimethyluridine);s4U(4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);ho5U(5-hydroxyuridine);mo5U(5-methoxyuridine);cmo5U(uridine 5-oxyacetic acid);mcmo5U(uridine 5-oxyacetic acid methyl ester);chm5U(5-(carboxyhydroxymethyl)uridine));mchm5U(5-(carboxyhydroxymethyl)uridine) mcm5U(5-methoxycarbonylmethyluridine);mcm5Um(S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine);nm5s2U(5-aminomethyl-2-thiouridine);mnm5U(5-methylaminomethyluridine);mnm5s2U(5-methylaminomethyl-2-thiouridine);mnm5se2U(5-methylaminomethyl-2-selenouridine);ncm5U(5-carbamoylmethyluridine);nc m5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine);cm5U (5-carboxymethyluridine); m6Am (N6,TO-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine); m'Am ((1,2-O-dimethyladenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine);or ac6A (N6-acetyl adenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine ... Cytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or abasic nucleotide. For example, the self-replicating RNA may include one or more modified pyrimidine nucleobases (e.g., pseudouridine and / or 5-methylcytosine residues). However, in some embodiments, the RNA does not contain modified nucleobases and may not contain modified nucleotides (i.e., all of the nucleotides in the RNA are standard A, C, G and U ribonucleotides (except for any 5' cap structure, which may include 7'-methylguanosine). In other embodiments, the RNA may contain a 5' cap that includes 7'-methylguanosine, and the first one, two or three 5' ribonucleotides may be methylated at the 2' position of the ribose;

[0038] The RNA used in the present invention ideally contains only phosphodiester linkages between nucleotides, although in some embodiments it may contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0039] Ideally, liposomes contain fewer than 10 different species of RNA (e.g., 5, 4, 3, or 2 different species); most preferably, liposomes contain a single RNA species (i.e., all RNA molecules in the liposome have the same sequence and length).

[0040] The amount of RNA per liposome can vary: the number of individual self-replicating RNAs per liposome is typically ≦50 (e.g., <20, <10, <5, or 1-4) per liposome.

[0041] (immunogen) The RNA molecules used in the present invention encode polypeptide immunogens. After administration of the liposomes, the RNA is translated in vivo and the immunogen can elicit an immune response in the recipient. The immunogen can elicit an immune response against bacteria, viruses, fungi or parasites (or, in some embodiments, against allergens; and in other embodiments, against tumor antigens). The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response. The polypeptide immunogens typically elicit an immune response that recognizes the corresponding bacterial, viral, fungal or parasitic (or allergen or tumor) polypeptide, although in some embodiments the polypeptides can act as mimotopes to elicit an immune response that recognizes saccharides of the bacteria, viruses, fungi or parasites. The immunogens are typically surface polypeptides (e.g., adhesins, hemagglutinins, envelope glycoproteins, spike glycoproteins, etc.).

[0042] The self-replicating RNA molecule may code for a single polypeptide immunogen or multiple polypeptides. Multiple immunogens may be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogens are expressed as separate polypeptides, one or more of them may be provided with an upstream IRES or additional viral promoter elements. Alternatively, multiple immunogens may be expressed from a polyprotein that codes for each immunogen fused to a short autocatalytic protease (e.g., foot-and-mouth disease virus 2A protein) or as an intein.

[0043] Unlike references 1 and 14, the RNA encodes an immunogen. For the avoidance of doubt, the present invention does not include RNA encoding firefly luciferase, RNA encoding a fusion protein of E. coli β-galactosidase, or RNA encoding green fluorescent protein (GFP). Also, the RNA is not whole mouse thymus RNA.

[0044] In some embodiments, the immunogen induces an immune response against one of these bacteria: Neisseria meningitidis: Useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins. Three useful polypeptide combinations are disclosed in reference 15.

[0045] Streptococcus pneumoniae: Useful polypeptide immunogens are disclosed in reference 16. These include, but are not limited to, the RrgB pilus subunit, β-N-acetyl-hexosaminidase precursor (spr0057), spr0096, the general stress protein GSP-781 (spr2021, SP2216), the serine / threonine kinase StkP (SP1732), and the pneumococcal surface adhesin PsaA.

[0046] Streptococcus pyogenes: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 17 and 18.

[0047] Moraxella catarrhalis Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous hemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.

[0048] Staphylococcus aureus: Useful immunogens include, but are not limited to, polypeptides disclosed in reference 19 (e.g., hemolysin, esxA, esxB, ferrichrome binding protein (sta006) and / or sta011 lipoprotein).

[0049] Clostridium tetani: The representative immunogen is tetanus toxoid.

[0050] Cornynebacterium diphtheriae: The representative immunogen is diphtheria toxoid.

[0051] Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 20 and 21.

[0052] Pseudomonas aeruginosa Streptococcus agalactiae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 17.

[0053] Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7 / L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g., as disclosed in reference 22). LcrE

[23] and HtrA

[24] are two preferred immunogens.

[0054] Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 25.

[0055] Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and / or urease

[26] .

[0056] Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and / or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and meningitis / septicemia associated E. coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in references 27 and 28. Useful MNEC immunogens are disclosed in reference 29. A useful immunogen for some E. coli types is AcfD

[30] .

[0057] Bacillus anthracis Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in references 31 and 32.

[0058] [ka]

[0059] [ka] In some embodiments, the immunogen induces an immune response against one of these viruses: Orthomyxovirus: Useful immunogens can be derived from influenza A, B, or C viruses (e.g., hemagglutinin, neuraminidase, or matrix M2 proteins). If the immunogen is an influenza A virus hemagglutinin, it can be from any subtype (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16).

[0060] Paramyxoviridae viruses: Viral immunogens include, but are not limited to, those derived from pneumonia viruses (e.g., respiratory syncytial virus, RSV), rubulaviruses (e.g., mumps virus), paramyxoviruses (e.g., parainfluenza virus), metapneumoviruses, and morbilliviruses (e.g., measles).

[0061] Poxviridae: Viral immunogens include, but are not limited to, those derived from orthopoxviruses, such as variola purpura (including, but not limited to, variola major and variola minor).

[0062] Picornavirus: Viral immunogens include, but are not limited to, those derived from picornaviruses, such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aphthoviruses. In one embodiment, the enterovirus is a poliovirus, such as polioviruses type 1, 2, and / or 3. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a Coxsackie A or B virus.

[0063] Bunyaviruses: Viral immunogens include, but are not limited to, those derived from an Orthobunyavirus (e.g., California encephalitis virus), a Phlebovirus (e.g., Rift Valley fever virus), or a Nairovirus (e.g., Crimean-Congo hemorrhagic fever virus).

[0064] Heparnavirus: Viral immunogens include, but are not limited to, those derived from a heparnavirus, such as Hepatitis A virus (HAV).

[0065] Filoviruses: Viral immunogens include, but are not limited to, those derived from a filovirus, such as an Ebola virus (including Zaire Ebola virus, Ivory Coast Ebola virus, Reston Ebola virus, or Sudan Ebola virus) or a Marburg virus.

[0066] Togavirus: Viral immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus, including a Rubella virus.

[0067] Flavivirus: Viral immunogens include, but are not limited to, those derived from a flavivirus (e.g., tick-borne encephalitis (TBE) virus, dengue (types 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur Forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus).

[0068] Pestivirus: Viral immunogens include, but are not limited to, those derived from a pestivirus, such as bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease virus (BDV).

[0069] Hepadnavirus: Viral immunogens include, but are not limited to, those derived from a hepadnavirus, such as Hepatitis B. The composition may include Hepatitis B surface antigen (HBsAg).

[0070] Other Hepatitis Viruses: The compositions may include immunogens derived from Hepatitis C virus, Hepatitis Delta virus, Hepatitis E virus, or Hepatitis G virus.

[0071] Rhabdoviruses: Viral immunogens include, but are not limited to, those derived from Rhabdoviruses, such as Lyssaviruses (eg, rabies virus) and Vesiculoviruses (VSV).

[0072] Caliciviridae: Viral immunogens include, but are not limited to, those derived from the Caliciviridae family, such as Norwalk virus (Norovirus), and Norwalk-like viruses, such as Hawaii virus and Snow Mountain virus.

[0073] Coronavirus: Viral immunogens include, but are not limited to, those derived from SARS coronavirus, infectious bronchitis avian virus (IBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis porcine virus (TGEV). The coronavirus immunogen may be a spike polypeptide.

[0074] Retroviruses: Viral immunogens include, but are not limited to, those derived from oncoviruses, lentiviruses (eg, HIV-1 or HIV-2), or spumaviruses.

[0075] Reovirus: Viral immunogens include, but are not limited to, those derived from an orthoreovirus, a rotavirus, an orbivirus, or a coltivirus.

[0076] Parvovirus: Viral immunogens include, but are not limited to, those derived from parvovirus B19.

[0077] Herpesviruses: Viral immunogens include, but are not limited to, those derived from human herpes viruses, such as, by way of example only, herpes simplex virus (HSV) (e.g., HSV types 1 and 2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV6), human herpes virus 7 (HHV7), and human herpes virus 8 (HHV8).

[0078] Papovavirus: Viral immunogens include, but are not limited to, those derived from papillomavirus and polyomavirus. The (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 (e.g., from one or more of serotypes 6, 11, 16 and / or 18).

[0079] Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36).

[0080] In some embodiments, the immunogen induces an immune response against a virus that infects fish (e.g., infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), piscine lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), carp herpesvirus, salmon picorna-like virus (also known as Atlantic salmon picorna-like virus), yamame virus (LSV), Atlantic salmon rotavirus (ASR), thrush disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV)).

[0081] Fungal immunogens include those from Dermatophytres, including:

[0082] [ka]

[0083] [ka] It can be derived from.

[0084] In some embodiments, the immunogen induces an immune response against a parasite from the genus Plasmodium (e.g., P. falciparum, P. vivax, P. malariae, or P. ovale). Thus, the present invention may be used to immunize against malaria. In some embodiments, the immunogen induces an immune response against a parasite from the family Caligidae, particularly a parasite from the genus Lepeophtheirus and the genus Caligus (e.g., a sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi).

[0085] In some embodiments, the immunogen induces an immune response against: pollen allergens (tree pollen, herbaceous pollen, weed pollen, and grass pollen allergens); insect or arachnid allergens (inhalation, saliva and venom allergens, e.g., mite allergens, cockroach allergens and midge allergens, hymenopthera venom allergens); animal hair and dander allergens (e.g., from dogs, cats, horses, rats, mice, etc.); and food allergens (e.g., gliadin). Important pollen allergens derived from trees, grasses and herbs are those derived from the taxonomic orders of Fagales, Oleales, Pinales and the family platanaceae (including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedars (Cryptomeria and Juniperus), plane tree (Platanus), the order of Poales (including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum), the orders Asterales and Urticales (including herbs of the genera Ambrosia, Artemisia, and Parietaria). Other important inhalant allergens are those from dust mites of the genera Dermatophagoides and Euroglyphus, storage mites (e.g. Lepidoglyphys, Glycyphagus and Tyrophagus), cockroaches, those from midges and fleas (e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides) and those from mammals (e.g. cats, dogs and horses), venom allergens (such as those from stinging or biting insects, including, for example, the taxonomic order Hymenoptera, including bees (Apidae), wasps (Vespidea) and ants (Formicoidae)).

[0086] In some embodiments, the immunogen is a tumor antigen selected from: (a) cancer-testis antigens, such as NY-ESO-1, SSX2, SCP1 and polypeptides of the RAGE, BAGE, GAGE ​​and MAGE families (e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12), which may be used to address, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, such as p53 (associated with various solid tumors, such as colorectal, lung, head and neck cancer), p21 / Ras (associated with melanoma, pancreatic and colorectal cancer), CDK4 (associated with melanoma), MUM1 (associated with melanoma), caspase-8 (associated with head and neck cancer), CIA 0205 (e.g., associated with bladder cancer), HLA-A2-R1701, β-catenin (e.g., associated with melanoma), TCR (e.g., associated with T cell non-Hodgkin's lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT 1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colon cancer), and carcinoembryonic antigen (e.g., associated with breast, lung, and gastrointestinal cancers, e.g., colorectal cancer);(d) shared antigens, e.g., melanoma-melanocyte differentiation antigens, e.g., MART-1 / Melan A, gp100, MC1R, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-related antigens (e.g., PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2 (e.g., associated with prostate cancer)); (f) immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens (including E6 and E7), antigens of hepatitis B virus and hepatitis C virus, human T-lymphotropic virus antigens;

[0087] [ka] (Mac-2 binding protein / cyclophilin C-related protein), TAAL6, TAG72, TLP, TPS, etc.

[0088] Pharmaceutical Compositions The liposomes of the present invention are useful as components in pharmaceutical compositions for immunizing subjects against various diseases. These compositions typically contain, in addition to the liposomes, a pharma- ceutically acceptable carrier. A detailed discussion of pharma-ceutically acceptable carriers is available in reference 33.

[0089] The pharmaceutical composition of the present invention may include one or more small molecule immune enhancing factors. For example, the composition may include a TLR2 agonist (e.g., Pam3CSK4), a TLR4 agonist (e.g., aminoalkyl glucosaminide phosphate (e.g., E6020)), a TLR7 agonist (e.g., imiquimod), a TLR8 agonist (e.g., resiquimod) and / or a TLR9 agonist (e.g., IC31). Any such agonist ideally has a molecular weight of <2000Da. When RNA is encapsulated, in some embodiments, such agonists are also encapsulated with the RNA, but in other embodiments, they are not encapsulated. When RNA is adsorbed to particles, in some embodiments, such agonists are also adsorbed with the RNA, but in other embodiments, they are not adsorbed.

[0090] The pharmaceutical composition of the present invention may contain the liposomes in plain water (e.g., WFI) or a buffer solution (e.g., phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer). Buffer salts are typically included in the range of 5 to 20 mM.

[0091] The pharmaceutical composition of the present invention may have a pH of 5.0 to 9.5 (eg, 6.0 to 8.0).

[0092] Compositions of the invention may include a sodium salt (e.g., sodium chloride) to provide tonicity. A concentration of 10±2 mg / ml NaCl is typical (e.g., about 9 mg / ml).

[0093] The compositions of the invention may include metal ion chelators. These may extend RNA stability by removing ions that may accelerate phosphodiester hydrolysis. Thus, the compositions may include one or more of EDTA, EGTA, BAPTA, pentetic acid, and the like. Such chelators are typically present at 10-500 μM (e.g., 0.1 mM). Citrates (e.g., sodium citrate) may also act as chelators while advantageously providing buffering activity.

[0094] The pharmaceutical composition of the present invention may have an osmolality of 200 mOsm / kg to 400 mOsm / kg (eg, 240 to 360 mOsm / kg, or 290 to 310 mOsm / kg).

[0095] Pharmaceutical compositions of the invention may contain one or more preservatives (e.g., thiomersal or 2-phenoxyethanol). Mercury-free compositions are preferred and preservative-free vaccines may be prepared.

[0096] Pharmaceutical compositions of the present invention are preferably sterile.

[0097] Pharmaceutical compositions of the invention are preferably non-pyrogenic (eg, containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose).

[0098] The pharmaceutical compositions of the present invention are preferably gluten free.

[0099] The pharmaceutical composition of the present invention may be prepared in a unit dose form. In some embodiments, the unit dose may have a volume of 0.1 to 1.0 ml (e.g., about 0.5 ml).

[0100] The compositions may be prepared as injections (either as solutions or suspensions). The compositions may be prepared for pulmonary administration (e.g., by inhaler) using a fine spray. The compositions may be prepared for administration to the nose, ear or eye (e.g., as a spray or drops). Injections for intramuscular administration are typical.

[0101] The composition contains an immunologically effective amount of liposomes, as well as any other components, if necessary. "Immunologically effective amount" means that the amount administered to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, the age of the individual to be treated, the taxonomic group (e.g., non-human primates, primates, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's evaluation of the medical situation, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined through routine clinical trials. The liposome and RNA content of the composition of the present invention is generally expressed in terms of the amount of RNA per dose. A preferred dose has ≦100 μg RNA (e.g., 10-100 μg (e.g., about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg)), although expression can be seen at much lower levels, e.g., ≦1 μg / dose, ≦100 ng / dose, ≦10 ng / dose, ≦1 ng / dose, etc.

[0102] The invention also provides a delivery device (e.g., a syringe, nebulizer, sprayer, inhaler, skin patch, etc.) containing a pharmaceutical composition of the invention, which can be used to administer the composition to a vertebrate subject.

[0103] The liposomes of the present invention do not contain ribosomes.

[0104] (Treatment Methods and Medical Uses) In contrast to the particles disclosed in reference 14, the liposomes and pharmaceutical compositions of the invention are intended for in vivo use to elicit an immune response against an immunogen of interest.

[0105] The present invention provides a method for eliciting an immune response in a vertebrate, comprising administering an effective amount of the liposome or pharmaceutical composition of the present invention.The immune response is preferably protective, and preferably includes antibodies and / or cell-mediated immunity.The method can elicit a booster response.

[0106] The invention also provides a liposome or pharmaceutical composition of the invention for use in a method for raising an immune response in a vertebrate.

[0107] The invention also provides the use of a liposome of the invention in the manufacture of a medicament for raising an immune response in a vertebrate.

[0108] By eliciting an immune response in the vertebrate by these uses and methods, the vertebrate can be protected from various diseases and / or infections (e.g., bacterial and / or viral diseases) as discussed above. The liposomes and compositions are immunogenic, and more preferably are vaccine compositions. Vaccines according to the present invention can be either prophylactic (i.e., prevent infection) or therapeutic (i.e., treat infection), but are typically prophylactic.

[0109] The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g., horse, cow, deer, goat, pig). If the vaccine is for prophylactic use, the human is preferably a child (e.g., a toddler or infant) or a teenager; if the vaccine is for therapeutic use, the human is preferably a teenager or an adult. Vaccines intended for children can also be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc.

[0110] The vaccine prepared according to the present invention can be used to treat both children and adults. Thus, the human patient may be under 1 year old, under 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients to receive the vaccine are elderly (e.g., ≧50 years old, ≧60 years old, and preferably ≧65 years old), young people (e.g., ≦5 years old), hospitalized patients, health care workers, military personnel and personnel, pregnant women, chronically ill patients, or immunocompromised patients. The vaccine is not only suitable for these groups, but can be used more generally in the population.

[0111] The compositions of the present invention are generally administered directly to patients. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of tissue; unlike Reference 1, intraglossal injection is not typically used in the present invention). Alternative delivery routes include rectal, oral (e.g., tablets, sprays), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection may be via a needle (e.g., a hypodermic needle), but needle-free injections can be used instead. A typical intramuscular dose is 0.5 ml.

[0112] The present invention may be used to induce systemic and / or mucosal immunity, preferably to induce enhanced systemic and / or mucosal immunity.

[0113] Dosage may be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule, various doses may be given by the same or different routes (e.g., parenteral primary and mucosal boost, mucosal primary and parenteral boost, etc.). Multiple doses are typically administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, multiple doses may be administered at about 6 weeks, 10 weeks, and 14 weeks of age (e.g., at 6 weeks, 10 weeks, and 14 weeks of age, as often used in the World Health Organization's Expanded Program on Immunisation ("EPI")). In an alternative embodiment, two primary doses are administered about two months apart (e.g., about seven, eight or nine weeks apart), followed by one or more booster doses about six months to one year after the second primary dose (e.g., about six, eight, ten or twelve months after the second primary dose). In a further embodiment, three primary doses are administered about two months apart (e.g., about seven, eight or nine weeks apart), followed by one or more booster doses about six months to one year after the third primary dose (e.g., about six, eight, ten or twelve months after the third primary dose).

[0114] (General) The particles of the present invention use, unless otherwise indicated, conventional methods within the skill of the art of chemistry, biochemistry, molecular biology, immunology and pharmacology. Such techniques are fully explained in the literature. See, for example, references 34-40.

[0115] The term "comprising" encompasses "including" as well as "consisting", e.g., a composition "comprising" X may consist exclusively of X or may include something additional (e.g., X+Y).

[0116] The term "about" in reference to a numerical value x is optional and means, for example, x±10%.

[0117] The term "substantially" does not exclude "completely", for example a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be omitted from the definition of the invention.

[0118] References to charges, cations, anions, zwitterions, etc. are addressed at pH 7.

[0119] TLR3 is Toll-like receptor 3. It is a single-pass transmembrane receptor that plays an important role in the innate immune system. Known TLR3 agonists include poly(I:C). "TLR3" is the accepted HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:11849. The RefSeq sequence of the human TLR3 gene is GI:2459625.

[0120] TLR7 is Toll-like receptor 7. It is a single-pass transmembrane receptor that plays an important role in the innate immune system. Known TLR7 agonists include, for example, imiquimod. "TLR7" is the accepted HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15631. The RefSeq sequence of the human TLR7 gene is GI:67944638.

[0121] TLR8 is Toll-like receptor 8. It is a single-pass transmembrane receptor that plays an important role in the innate immune system. Known TLR8 agonists include, for example, resiquimod. "TLR8" is the accepted HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15632. The RefSeq sequence of the human TLR8 gene is GI:20302165.

[0122] The RIG-I-like receptor ("RLR") family includes various RNA helicases that play important roles in the innate immune system

[41] . RLR-1 (also known as RIG-I or retinoic acid-inducible gene I) has two caspase recruitment domains near its N-terminus. The accepted HGNC name for the gene encoding the RLR-1 helicase is "DDX58" (for DEAD (Asp-Glu-Ala-Asp) box polypeptide 58) and its unique HGNC ID is HGNC:19102. The RefSeq sequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known as MDA5 or melanoma differentiation-associated gene 5) also has two caspase recruitment domains near its N-terminus. The accepted HGNC name of the gene encoding the RLR-2 helicase is "IFIH1" (for interferon induced with helicase C domain 1) and the unique HGNC ID is HGNC:18873. The RefSeq sequence of the human RLR-2 gene is GI:27886567. RLR-3 (also known as LGP2 or laboratory of genetics and physiology 2) does not have a caspase recruitment domain. The accepted HGNC name of the gene encoding the RLR-3 helicase is "DHX58" (for DEXH (Asp-Glu-X-His) box polypeptide 58) and the unique HGNC ID is HGNC:29517. The RefSeq sequence of the human RLR-3 gene is GI:149408121.

[0123] PKR is a double-stranded RNA-dependent protein kinase. It plays an important role in the innate immune system. "EIF2AK2" (for eukaryotic translation initiation factor 2-alpha kinase 2) is the accepted HGNC name of the gene encoding this enzyme, and its unique HGNC ID is HGNC:9437. The RefSeq sequence of the human PKR gene is GI:208431825. [Brief description of the drawings]

[0124] [Figure 1] Figure 1 shows a gel with stained RNA. The lanes show: (1) marker, (2) naked replicon, (3) replicon after RNase treatment, (4) replicon encapsulated in liposomes, (5) liposomes after RNase treatment, (6) liposomes treated with RNase and then subjected to phenol / chloroform extraction. [Diagram 2] FIG. 2 is an electron microscope photograph of liposomes. [Diagram 3] FIG. 3 shows the structures of DLinDMA, DLenDMA and DODMA. [Figure 4] Figure 4 shows a gel with stained RNA. The lanes show (1) marker, (2) naked replicon, (3) replicon encapsulated in liposomes, and (4) liposomes treated with RNase and then subjected to phenol / chloroform extraction. [Diagram 5] FIG. 5 shows protein expression at days 1, 3, and 6 after delivery of RNA as a virion-packaged replicon (squares), as naked RNA (diamonds), or in liposomes (+=0.1 μg, ×=1 μg). [Figure 6] FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA. [Figure 7]FIG. 7 shows anti-F IgG titers in animals given virion packaged replicons (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulated RNA. [Figure 8] FIG. 8 shows anti-F IgG titers in animals given VRP, 1 μg naked RNA, and 0.1 g or 1 μg liposome-encapsulated RNA. [Figure 9] FIG. 9 shows neutralizing antibody titers in animals given either VRP, or 0.1 g or 1 μg of liposome-encapsulated RNA. [Figure 10] FIG. 10 shows expression levels following delivery of the replicon as naked RNA (circles), liposome-encapsulated RNA (triangles and squares), or as a lipoplex (inverted triangles). [Figure 11] FIG. 11 shows F-specific IgG titers (2 weeks after the second dose) after delivery of the replicon as naked RNA (0.01-1 μg), as liposome-encapsulated RNA (0.01-10 μg), or packaged as virions (VRP, 10 infectious units or IU). [Figure 12] Figure 12 shows F-specific IgG titers (circles) and PRNT titers (squares) after delivery of the replicon as naked RNA (1 μg), as liposome-encapsulated RNA (0.1 or 1 μg), or packaged as virions (VRP, 106 IU). Titers in naive mice are also shown. Solid lines indicate the geometric mean. [Figure 13] Figure 13 shows intracellular cytokine production after restimulation with synthetic peptides representing major epitopes in the F protein 4 weeks after the second dose. The y-axis shows % cytokine+ of CD8+CD4-. [Figure 14A] Figure 14 shows F-specific IgG titers (mean log10 titers ± standard deviation) over 63 days (Figure 14A) and 210 days (Figure 14B) after immunization of calves. Three lines are easily distinguishable at day 63, from bottom to top: PBS negative control; liposomally delivered RNA; and the "Triangle 4" product. [Figure 14B] Figure 14 shows F-specific IgG titers (mean log10 titers ± standard deviation) over 63 days (Figure 14A) and 210 days (Figure 14B) after immunization of calves. Three lines are easily distinguishable at day 63, from bottom to top: PBS negative control; liposomally delivered RNA; and the "Triangle 4" product. [Figure 15] Figure 15 shows SEAP expression (relative intensity) on day 6 versus the pKa of the lipid used in the liposomes. Circles indicate levels for liposomes with DSPC and squares indicate levels for liposomes without DSPC; sometimes the squares and circles overlap, making only the square visible for a given pKa. [Figure 16] Figure 16 shows the expression of anti-F titers (relative to RV01, 100%) two weeks after the first administration of a replicon encoding the F protein. The titers are plotted against pKa in the same manner as in Figure 15. The asterisks indicate RV02, which used a cationic lipid with a higher pKa than the other lipids. The triangles indicate data for liposomes lacking DSPC; the circles are data for liposomes that included DSPC. [Figure 17] Figure 17 shows total IgG titers after replicon delivery in liposomes using, from left to right, RV01, RV16, RV17, RV18 or RV19. Bars show mean values. The upper bar in each case is 2wp2 (i.e., 2 weeks after the second dose) whereas the lower bar is 2wp1. [Figure 18] Figure 18 shows IgG titers in 13 groups of mice. Each circle is an individual mouse and the solid line indicates the geometric mean. The horizontal dashed line is the detection limit of the assay. The 13 groups are, from left to right, A to M as shown below. [Figure 19]Figure 19 shows (A) IL-6 and (B) IFNα (pg / ml) released by pDC. There are four pairs of bars, from left to right: control; immunogenic with RNA + DOTAP; immunogenic with RNA + lipofectamine; and immunogenic with RNA in liposomes. In each pair, the black bar is wild type mouse and the grey is rsq1 mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0125] (RNA replicon) Various replicons are used below. Generally, they are based on a hybrid alphavirus genome with nonstructural proteins from Venezuelan Equine Encephalitis Virus (VEEV), a packaging signal from Sindbis Virus, and a 3'UTR from Sindbis Virus or a VEEV variant. The replicons are approximately 10 kb long and have a polyA tail.

[0126] Plasmid DNA encoding an alphavirus replicon (designated pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for in vivo RNA synthesis. The replicons contain the alphavirus genetic elements required for RNA replication but lack elements encoding gene products required for particle assembly; the structural proteins are instead replaced by proteins of interest, either reporters (e.g., SEAP or GFP) or immunogens (e.g., full-length RSV F protein), and the replicons are unable to direct the production of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA drives the synthesis of the replicon RNA in vitro, and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A) tail generates precise 3' ends via its self-cleavage activity.

[0127] After linearizing the plasmid DNA downstream of the HDV ribozyme with the appropriate restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage-derived DNA-dependent RNA polymerase. Transcription was carried out for 2 hours at 37°C in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP, and UTP) according to the instructions provided by the manufacturer (Ambion). After transcription, the template DNA was digested with TURBO DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was post-transcriptionally capped with Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G Capping System (Epicentre Biotechnologies) as outlined in the user manual; replicons capped in this way are prefixed with the letter "v" (e.g., vA317 is an A317 replicon capped by VCE). Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentrations of the RNA samples were determined by OD 260nm The integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis.

[0128] (Liposome encapsulation) RNA was encapsulated in liposomes made by the methods of references 11 and 42. The liposomes were made of 10% DSPC (zwitterionic), 40% DLinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These percentages refer to % moles in the total liposomes.

[0129] DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure in reference 6. DSPC (1,2-diastearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids.

[0130] Briefly, lipids were dissolved in ethanol (2 ml), and RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6), which were mixed with 2 ml of buffer, followed by equilibration for 1 hour. The mixture was diluted with 6 ml of buffer, and then filtered. The resulting product contained liposomes with an encapsulation efficiency of about 95%.

[0131] For example, in one particular method, a fresh lipid stock solution was prepared in ethanol. 37 mg DLinDMA, 11.8 mg DSPC, 27.8 mg cholesterol, and 8.07 mg PEG-DMG were weighed and dissolved in 7.55 mL ethanol. The freshly prepared lipid stock solution was gently shaken at 37° C. for about 15 minutes to form a homogenous mixture. 755 μL of the stock was then added to 1.245 mL ethanol to make 2 mL of working lipid stock solution. This amount of lipid was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of about 1 μg / μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with copious amounts of MilliQ water before use to remove RNase contamination from the vials. One of the vials was used for the RNA working solution and the other was used to collect the lipid and RNA mixture (as described later). The working lipid and RNA solutions were heated at 37°C for 10 minutes and then placed into a 3cc Luer lock syringe. 2mL citrate buffer (pH 6) was placed into another 3cc syringe. The syringe containing the RNA and lipid was then inserted into a T-mixer (PEEK) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used had a 2mm inner diameter and a 3mm outer diameter). TMThe first syringe was connected to a 500 μm ID junction (Idex Health Science). The outlet from the T-mixer was also FEP tubing. A third syringe containing the citrate buffer was connected to a separate piece of FEP tubing. All syringes were then run using a syringe pump at a flow rate of 7 mL / min. The tubing outlet was positioned to collect the mixture in a 20 mL glass vial (with stirring). The stir bar was removed and the ethanol / aqueous solution was allowed to equilibrate to room temperature for 1 hour. 4 ml of the mixture was placed in a 5 cc syringe, which was connected to one of the FEP tubings, and another 5 cc syringe was connected to an equal length of FEP tubing and contained an equal amount of 100 mM citrate buffer (pH 6). The two syringes were run using the syringe pump at a flow rate of 7 mL / min and the final mixture was collected in a 20 mL glass vial (with stirring). The mixture collected from the second mixing step (liposomes) was then passed through a Mustang Q membrane (anion exchange support obtained from Pall Corporation, which binds and removes anionic molecules). Prior to using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl, and 10 mL of 100 mM citrate buffer (pH 6) were passed through the membrane in succession. The liposomes were warmed at 37°C for 10 minutes and then passed through the membrane. Next, the liposomes were concentrated to 2 mL by using tangential flow filtration and dialyzed against 10-15 volumes of 1x PBS, after which the final product was collected. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and used according to the manufacturer's guidelines. The 100 kD pore size cutoff and 8 cm 2 A polysulfone hollow fiber filtration membrane with a surface area of ​​100 μg / ml was used. For in vitro and in vivo experiments, the formulations were diluted with 1×PBS to the required RNA concentration.

[0132] 2 shows exemplary electron micrographs of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding the full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number).

[0133] The percentage of encapsulated RNA and the concentration of RNA were determined by Quant-iT RiboGreen RNA Reagent Kit (Invitrogen) according to the manufacturer's instructions. A standard curve was generated using the ribosomal RNA standard provided in the kit. Liposomes were diluted 10x or 100x in 1x TE buffer (from the kit) before dye was added. Separately, liposomes were diluted 10x or 100x in 1x TE buffer containing 0.5% Triton X before dye was added (to disrupt the liposomes and thus assay total RNA). An equal amount of dye was then added to each solution, and then about 180 μL of each solution after dye addition was placed in duplicate into a 96-well tissue culture plate. Fluorescence (excitation 485 nm, emission 528 nm) was read on a microplate reader. All liposome formulations were administered in vivo based on the amount of encapsulated RNA.

[0134] Encapsulation in liposomes has been shown to protect RNA from RNase digestion. Experiments used 3.8mAU RNase A / μg RNA and incubated at room temperature for 30 minutes. RNase was inactivated with proteinase K at 55°C for 10 minutes. The RNA was then extracted from the lipids into the aqueous phase by adding a 1:1 v / v mixture of phenol:chloroform:isoamyl alcohol to the sample 25:24:1 v / v / v. Samples were mixed by vortexing for a few seconds and then placed in a centrifuge at 12k RPM for 15 minutes. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Before loading (400ng RNA / well), all the samples were incubated with formaldehyde loading dye, denatured at 65°C for 10 minutes, and cooled to room temperature. The molecular weight of the RNA constructs was estimated using Ambion Millennium markers. The gel was run at 90V. The gel was stained using 0.1% SYBR Gold in water according to the manufacturer's guidelines by shaking at room temperature for 1 h. Figure 1 shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4) and no change is observed when the liposomes are treated with RNase (lane 4). After subjecting the RNase-treated liposomes to phenol extraction, undigested RNA is visible (lane 6). Even after one week at 4°C, the RNA could be seen without any fragmentation (Figure 4, arrow). In vivo protein expression was unchanged after six weeks at 4°C and one freeze-thaw cycle. Thus, liposome-encapsulated RNA is stable.

[0135] To assess in vivo expression of the RNA, a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon rather than an immunogen. Expression levels were measured in serum diluted 1:4 in 1x Phospha-Light dilution buffer using a chemiluminescent alkaline phosphatase substrate. 8-10 week old BALB / c mice (5 per group) were injected intramuscularly with 50 μl / leg on day 0 at 0.1 μg or 1 μg RNA doses. The same vector was also administered without the liposomes at 1 μg (in RNase-free 1x PBS). Virion packaged replicons were also tested. The virion packaged replicons (referred to as "VRP") used herein were obtained by the method of reference 43. Here, alphavirus replicons, either derived from mutant VEEV or chimeras derived from the genome of VEEV engineered to contain the Sindbis virus 3'UTR and Sindbis virus packaging signal (PS), were packaged by co-electroporation into BHK cells with defective helper RNAs encoding Sindbis virus capsid and glycoprotein genes.

[0136] As shown in Figure 5, encapsulation increased SEAP levels by approximately 1 / 2 log at the 1 μg dose, and at day 6, expression from the 0.1 μg encapsulated dose was comparable to that seen with the 1 μg unencapsulated dose. By day 3, expression levels exceeded those achieved with VRP (squares). Thus, expression was increased when the RNA was formulated in the liposomes, even at a 10x lower dose compared to the naked RNA control. Expression was also higher compared to the VRP control, but the kinetics of expression were very different (see Figure 5). Delivery of the RNA using electroporation resulted in increased expression compared to the naked RNA control, but these levels were lower than with liposomes.

[0137] To assess whether the effects seen in the liposome groups were due solely to the components of the liposomes or were related to the encapsulation, the replicon was administered in encapsulated form (2 different purification protocols, 0.1 μg RNA), mixed with the liposomes after their formation (non-encapsulated "lipoplex", 0.1 μg RNA), or as naked RNA (1 μg). Figure 10 shows that the lipoplex gave the lowest level of expression, indicating that encapsulation is essential for robust expression.

[0138] In vivo studies using liposome delivery confirmed these findings. Mice were given various combinations of (i) a self-replicating RNA replicon encoding the full-length RSV F protein, (ii) a self-replicating GFP-encoding RNA replicon, (iii) a GFP-encoding RNA replicon with a knockout in nsP4 that abolishes self-replication, and (iv) a full-length RSV F protein. A total of 13 groups were given:

[0139] [Table 1] The results in Figure 18 show that F-specific IgG responses required encapsulation in the liposomes rather than simple co-delivery (compare groups C and D). Comparison of groups K, L and M shows that the RNA provided an adjuvant effect to the co-delivered protein, an effect that was seen with both replicating and non-replicating RNA.

[0140] Further SEAP experiments showed a clear dose response in vivo. Expression was observed after delivery of as little as 1 ng of RNA (Figure 6). Further experiments comparing expression from encapsulated replicons to naked replicons showed that 0.01 μg encapsulated RNA was equivalent to 1 μg naked RNA. At a 0.5 μg dose of RNA, the encapsulated material gave 12-fold higher expression at day 6; at the 0.1 μg dose level, it was 24-fold higher at day 6.

[0141] In addition to examining the average levels in the groups, individual animals were also studied. Some animals were non-responders to the naked replicon, whereas encapsulation eliminated non-responders.

[0142] In further experiments, DLinDMA was replaced with DOTAP. Although DOTAP liposomes gave better expression than naked replicons, they were inferior to DLinDMA liposomes (2-3 fold difference at day 1). DOTAP has a quaternary amine and therefore a positive charge in terms of delivery, whereas DLinDMA has a tertiary amine.

[0143] To assess immunogenicity in vivo, replicons were constructed to express full-length F protein from respiratory syncytial virus (RSV), either naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (10 6 IU; "VRP") were delivered on days 0 and 21. Figure 7 shows anti-F IgG titers two weeks after the second dose. The liposomes clearly enhance immunogenicity. Figure 8 shows the titers after two weeks. By this time point, there was no statistical difference between the 0.1 μg encapsulated RNA, the 1 μg encapsulated RNA, and the VRP groups. Neutralization titers (measured as 60% plaque reduction, "PRNT60") were not significantly different in the three groups two weeks after the second dose (Figure 9). Figure 12 shows both IgG and PRNT titers four weeks after the second dose.

[0144] FIG. 13 confirms that the RNA induces robust CD8 T cell responses.

[0145] In further experiments, F-specific IgG titers were compared in mice given VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. The titer ratios (VRP:liposome) at various time points after the second dose were as follows:

[0146] [Table 2] Thus, the liposome-encapsulated RNA induces an immune response essentially similar to that seen with virion delivery.

[0147] Further experiments showed a good F-specific IgG response at the 10 μg dose, which was equivalent to the responses for the 1 μg and 0.1 μg doses, with a lower response at the 0.01 μg dose. Figure 11 shows the results of F-specific IgG responses in naked form at three different doses, in liposomes at four different doses, or in VRP (10 6 IgG titers in animals given the replicon are shown as IU. The response observed with 1 μg liposome-encapsulated RNA was not statistically significant when compared to VRP (ANOVA), but the higher response observed with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups.

[0148] Further studies showed that the 0.1 μg liposome-encapsulated RNA gave a much higher anti-F IgG response (15 days after the second dose) than 0.1 μg of delivered DNA, and furthermore, electroporation (Elgen TM The F antigen was confirmed to be immunogenic by 20 μg plasmid DNA encoding the F antigen delivered by the Inovio DNA Delivery System.

[0149] Further studies were performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose, liposome encapsulation increased F-specific IgG titers 8.3-fold and PRNT titers 9.5-fold compared to naked RNA. The magnitude of the antibody response was 5×10 6 Both naked RNA and liposome-encapsulated RNA were equivalent to those induced by RSV challenge (1 × 10 5 The antibody was able to protect the cotton rats from pulmonary viral load (PFU) and reduced lung viral load by at least 3.5 logs. Encapsulation enhanced the reduction by approximately 2-fold.

[0150] A large animal study was conducted in cattle. Cattle were immunized with 66 μg of the replicon encoding the full-length RSV F protein formulated inside liposomes on days 0 and 21. PBS alone was used as a negative control, and the approved vaccine was used as a positive control (Fort Dodge's "Triangle 4", containing killed virus). Figure 14 shows F-specific IgG titers over a 63-day period starting from the first immunization. The RNA replicon was immunogenic in cattle, but gave lower titers than the approved vaccine. All vaccinated cattle showed F-specific antibodies after the second dose, and titers were very stable (and especially stable for the RNA vaccine) over a period of 2 to 6 weeks after the second dose.

[0151] (Mechanism of action) Bone marrow-derived dendritic cells (pDCs) were obtained from wild-type mice or from a “Resq” (rsq1) mutant line, which has a point mutation in the amino terminus of the TLR7 receptor that abolishes TLR7 signaling without affecting ligand binding

[44] . The cells were stimulated with DOTAP, replicon RNA formulated with Lipofectamine 2000, or replicon RNA in liposomes. As shown in Figure 19, IL-6 and INFα were induced in WT cells, but this response was almost completely abolished in mutant mice. These results indicate that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferon and pro-inflammatory cytokines.

[0152] (pKa measurement) The pKa of lipids is measured in water at standard temperature and pressure using the following technique: · Prepare a 2 mM solution of lipids in ethanol by weighing the lipids and dissolving them in ethanol. Prepare a 0.3 mM solution of the fluorescent probe toluene nitrosulfonic acid (TNS) in ethanol:methanol 9:1 by first making a 3 mM solution of TNS in methanol and then diluting to 0.3 mM with ethanol. Prepare an aqueous buffer solution containing sodium phosphate, sodium citrate, sodium acetate, and sodium chloride at concentrations of 20 mM, 25 mM, 20 mM, and 150 mM, respectively. Divide the buffer into eight portions and adjust the pH to 4.44-4.52, 5.27, 6.15-6.21, 6.57, 7.10-7.20, 7.72-7.80, 8.27-8.33, and 10.47-11.12 with either 12N HCl or 6N NaOH. Mix 400 μL of 2 mM lipid solution and 800 μL of 0.3 mM TNS solution. · Add 7.5 μL of the probe / lipid mixture to 242.5 μL of buffer in a 1 mL 96-well plate. Do this for all eight buffers. After mixing, transfer 100 μL of each probe / lipid / buffer mixture to a clear bottom 250 μL black 96-well plate (e.g., model COSTAR 3904, Corning). A convenient way to perform this mixing is to use a Tecan Genesis RSP150 high-throughput liquid handler and Gemini Software. Measure the fluorescence of each probe / lipid / buffer mixture at 322 nm excitation, 431 nm emission (autocutoff 420 nm) (e.g., with a SpectraMax M5 spectrophotometer and SoftMax pro 5.2 software). After measurement, the background fluorescence value of empty wells in the 96-well plate is subtracted from each probe / lipid / buffer mixture. The fluorescence intensity values ​​are then normalized to the value at the lowest pH. The normalized fluorescence intensity is then plotted against pH to provide a line of best fit. Find the point on the line of best fit where the normalized fluorescence intensity is equal to 0.5. Find the pH that corresponds to a normalized fluorescence intensity equal to 0.5 and consider this the pKa of the lipid.

[0153] This method gives a pKa of 5.8 for DLinDMA. The pKa values ​​determined by this method for the cationic lipids of reference 5 are included below.

[0154] Encapsulation in Liposomes Using Alternative Cationic Lipids As an alternative to using DlinDMA, the cationic lipids of reference 5 are used. These lipids may be synthesised as disclosed in reference 5.

[0155] The liposomes formed above using DlinDMA are hereafter referred to as the "RV01" series. The DlinDMA was replaced with various cationic lipids as described below in the series "RV02"-"RV12". Two different types of each liposome were formed using 2% PEG2000-DMG and either (01) 40% of the cationic lipid, 10% DSPC, and 48% cholesterol, or (02) 60% of the cationic lipid and 38% cholesterol. Thus, a comparison of (01) and (02) liposomes shows the effect of neutral zwitterionic lipids.

[0156] RV02 liposomes were made using the following cationic lipids (pKa>9, no tertiary amines):

[0157] [ka] RV03 liposomes were made using the following cationic lipids (pKa 6.4):

[0158] [ka] RV04 liposomes were made using the following cationic lipids (pKa 6.62):

[0159] [ka] RV05 liposomes were made using the following cationic lipids (pKa 5.85):

[0160] [ka] RV06 liposomes were made using the following cationic lipids (pKa 7.27):

[0161] [ka] RV07 liposomes were made using the following cationic lipids (pKa 6.8):

[0162] [ka] RV08 liposomes were made using the following cationic lipids (pKa 5.72):

[0163] [ka] RV09 liposomes were made using the following cationic lipids (pKa 6.07):

[0164] [ka] RV10 liposomes were made for comparison using the following cationic lipid (pKa 7.86):

[0165] [ka] RV11 liposomes were made using the following cationic lipid (pKa 6.41):

[0166] [ka] RV12 liposomes were made using the following cationic lipids (pKa 7):

[0167] [ka] RV16 liposomes were made using the following cationic lipids (pKa 6.1)

[45] :

[0168] [ka] RV17 liposomes were made using the following cationic lipids (pKa 6.1)

[45] :

[0169] [ka] RV18 liposomes were made using DODMA, RV19 liposomes were made using DOTMA, and RV13 liposomes were made with DOTAP, both of which have a quaternary amine head group.

[0170] These liposomes were characterized and tested with the SEAP reporter described above. The table below shows the size of the liposomes (Z-average and polydispersity index), the % of RNA encapsulation in each liposome, and the SEAP activity detected at 1 and 6 days after injection. SEAP activity is related to "RV01(02)" liposomes made from DLinDMA, cholesterol and PEG-DMG:

[0171] [Table 3] Figure 15 plots SEAP levels against the pKa of the cationic lipid on day 6. Best results are seen when the lipid has a pKa between 5.6-6.8, and ideally, between 5.6-6.3.

[0172] These liposomes were also used to deliver a replicon encoding the full-length RSV F protein. Total IgG titers against the F protein two weeks after the first dose (2wp1) are plotted against pKa in Figure 16. Best results are seen when the pKa of the cationic lipid is between 5.7 and 5.9, but pKa alone is not sufficient to guarantee high titers (e.g., the lipid must still support liposome formation). (RSV immunogenicity) Further studies were performed with a self-replicating replicon (vA317) encoding the RSV F protein. BALB / c mice (4 or 8 animals per group) were vaccinated bilaterally intramuscularly (50 μL / leg) on ​​days 0 and 21 with the replicon (1 μg) alone or with the replicon formulated as liposomes with the RV01 or RV05 lipids (see above; pKa 5.8 or 5.85), or with RV13. The RV01 liposomes had 40% DLinDMA, 10% DSPC, 48% cholesterol, and 2% PEG-DMG, but with different amounts of RNA. The RV05(01) liposomes had 40% cationic lipid, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; the RV05(02) liposomes had 60% cationic lipid, 38% cholesterol, and 2% PEG-DMG. The RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol, and 2% PEG-DMG. For comparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigen was delivered either using electroporation or by RV01(10) liposomes (0.1 μg DNA). Four mice were used as a naive control group.

[0173] Liposomes were prepared by method (A) or method (B). In method (A), a fresh lipid stock solution in ethanol was prepared. 37 mg cationic lipid, 11.8 mg DSPC, 27.8 mg cholesterol and 8.07 mg PEG-DMG were weighed and dissolved in 7.55 mL ethanol. The freshly prepared lipid stock solution was gently shaken at 37° C. for about 15 minutes to form a homogenous mixture. Then, 226.7 μL of the above stock was added to 1.773 mL ethanol to make 2 mL of working lipid stock solution. This amount of lipid was used to form liposomes with 75 μg RNA, giving an 8:1 nitrogen to phosphate ratio (except in RV01(08) and RV01(09), this ratio was modified to 4:1 or 16:1). A 2 mL working solution of RNA (or DNA for RV01(10)) was also prepared from a stock solution of approximately 1 μg / μL in 100 mM citrate buffer, pH 6. Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with copious amounts of MilliQ water before use to remove RNase contamination from the vials. One of the vials was used for the RNA working solution and the other was used to collect the lipid and RNA mixture (as described below). The working lipid and RNA solutions were heated at 37°C for 10 minutes and then loaded into a 3 cc syringe. 2 mL of citrate buffer, pH 6, was loaded into another 3 cc syringe. The syringe containing the RNA and the lipids was then loaded into a T-mixer (PEEK 1000 mL) using FEP tubing. TMA 5 cc syringe was connected to a 500 μm ID junction (500 μm ID junction). The outlet from the T-mixer was also FEP tubing. A third syringe containing the citrate buffer was connected to a separate section of FEP tubing. All syringes were then run at a flow rate of 7 mL / min using a syringe pump. The outlet of the tubing was positioned to collect the mixture (with stirring) in a 20 mL glass vial. The stir bar was removed and the ethanol / aqueous solution was allowed to equilibrate to room temperature for 1 hour. The mixture was then loaded into a 5 cc syringe attached to a section of FEP tubing, and another 5 cc syringe with an equal length of FEP tubing was loaded with an equal volume of 100 mM citrate buffer (pH 6). The two syringes were run at a flow rate of 7 mL / min using a syringe pump and the final mixture was collected (with stirring) in a 20 mL glass vial. The liposomes were then concentrated to 2 mL and dialyzed against 10-15 volumes of 1x PBS using TFF, after which the final product was collected. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs and used according to the manufacturer's guidelines. The 100 kD pore size cutoff and 20 cm 2 A polyethersulfone (PES) hollow fiber filtration membrane with surface area (part number P-C1-100E-100-01N) was used. For in vitro and in vivo experiments, the formulations were diluted to the required RNA concentration in 1×PBS.

[0174] Preparation method (B) differs from method (A) in two ways. First, after collection in the 20 mL glass vial (but before TFF concentration), the mixture was passed through a Mustang Q membrane (an anion exchange support that binds and removes anionic molecules, obtained from Pall Corporation, Ann Arbor, MI, USA). The membrane was first washed with 4 mL of 1 M NaOH, then with 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6), and the liposomes were warmed at 37° C. for 10 minutes and then filtered. Second, the hollow fiber filtration membrane was polysulfone (part number P / N: X1AB-100-20P).

[0175] The Z-average particle diameter, polydispersity index, and encapsulation efficiency of the liposomes were as follows:

[0176] [Table 5] Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were harvested from mice on day 49 for T cell analysis.

[0177] F-specific serum IgG titers (GMTs) were as follows:

[0178] [Table 6] The percentage of T cells that were cytokine positive and specific for the RSV F51-66 peptide was as follows, with only numbers statistically significantly above zero shown:

[0179] [Table 7] Thus, the liposomal formulation significantly increased immunogenicity compared to the naked RNA control as determined by increased F-specific IgG titers and T cell frequencies. Plasmid DNA formulated with liposomes or delivered naked using electroporation was significantly less immunogenic than liposomally formulated self-replicating RNA.

[0180] The RV01 and RV05 RNA vaccines were more immunogenic than the RV13 (DOTAP) vaccine. The formulations have comparable physical characteristics and were formulated with the same self-replicating RNA, but they contain different cationic lipids. RV01 and RV05 both have tertiary amines in the head group with pKa ≈5.8 and also contain unsaturated alkyl tails. RV13 has an unsaturated alkyl tail, but its head group has a quaternary amine and is very cationic. These results suggest that lipids with tertiary amines, with pKa in the range of 5.0-7.6, are superior to lipids such as DOTAP, which are very cationic when used in liposomal delivery systems for RNA.

[0181] (Further alternative to DLinDMA) The cationic lipid (DLinDMA) in RV01 liposomes was replaced with RV16, RV17, RV18 or RV19. Total IgG titers are shown in Figure 17. The lowest results are seen with RV19 (i.e., DOTMA quaternary amine).

[0182] (BHK expression) Liposomes with different lipids were incubated overnight with BHK cells and evaluated for protein expression efficacy. From baseline with RV05 lipid expression could be increased 18× by adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposomes, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× by using RV01 instead.

[0183] RSV immunogenicity in various mouse strains The replicon "vA142" encodes the full-length wild-type surface fusion (F) glycoprotein of RSV, but with its fusion peptide deleted and its 3' end formed by ribozyme-mediated cleavage, and was tested in three different mouse strains.

[0184] BALB / c mice were given bilateral intramuscular vaccinations (50 μL / leg) on ​​days 0 and 22. Animals were divided into eight test groups (5 animals per group) and naive controls (2 animals): Group 1 received the naked replicon (1 μg). Group 2 received 1 μg replicon delivered in liposomes “RV01 (37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-conjugated DMG. Group 3 received the same as group 2, but with 0.1 μg RNA. Group 4 received 1 μg replicon in "RV17(10)" liposomes (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG). Group 5 received 1 μg replicon in "RV05(11)" liposomes (40% RV07 lipid, 30% 18:2 PE (DLoPE), 28% cholesterol, 2% PEG-DMG). Group 6 received 0.1 μg replicon in "RV17(10)" liposomes. Group 7 received 5 μg RSV-F subunit protein adjuvanted with aluminum hydroxide. Group 8 was naive control (2 animals).

[0185] Serum was collected for antibody analysis on days 14, 35 and 49. F-specific serum IgG GMTs were:

[0186] [Table 8] On day 35, F-specific IgG1 and IgG2a titers (GMT) were as follows:

[0187] [Table 9] RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers of 2-5 mouse pools (1 pool per group)):

[0188] [Table 10] On day 49, spleens were harvested for T cell analysis. The mean net F-specific cytokine positive T cell frequencies (CD4+ or CD8+) that were statistically significantly above zero (specific for RSV peptides F51-66, F164-178, F309-323 for CD4+, or peptides F85-93 and F249-258 for CD8+) were as follows (numbers only shown):

[0189] [Table 11] C57BL / 6 mice were immunized in the same manner, except that the ninth group received immunization with VRP (1 × 10 6 IU).

[0190] Serum was collected for antibody analysis on days 14, 35 and 49. F-specific IgG titers (GMT) were:

[0191] [Table 12] On day 35, F-specific IgG1 and IgG2a titers (GMT) were as follows:

[0192] [Table 13] RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers of 2-5 mouse pools (1 pool per group)):

[0193] [Table 14] On day 49, spleens were harvested for T cell analysis. The mean net F-specific cytokine positive T cell frequencies (CD8+) were as follows (only numbers that were statistically significantly above zero (specific for RSV peptides F85-93 and F249-258) are shown):

[0194] [Table 15] Nine groups of C3H / HeN mice were immunized in the same manner. F-specific IgG titers (GMTs) were as follows:

[0195] [Table 16] On day 35, F-specific IgG1 and IgG2a titers (GMT) were as follows:

[0196] [Table 17] RSV serum neutralizing antibody titers on days 35 and 49 were as follows:

[0197] [Table 18] Thus, three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) were tested in three different inbred mouse strains. For all three strains, RV01 was more effective than RV17; for BALB / c and C3H strains, RV05 was less effective than RV01 and RV17, but more effective in the B6 strain. However, in all cases, the liposomes were more effective than two cationic nanoemulsions tested in parallel.

[0198] (CMV immunogenicity) RV01 liposomes with DLinDMA as the cationic lipid were used to deliver RNA replicons encoding cytomegalovirus (CMV) glycoproteins. The "vA160" replicon encodes full-length glycoproteins H and L (gH / gL), whereas the "vA322" replicon encodes the soluble forms (gHsol / gL). The two proteins are under the control of separate subgenomic promoters in a single replicon; co-administration of two separate vectors (one encoding gH and one encoding gL) did not give good results.

[0199] BALB / c mice (10 mice / group) were inoculated with gH / gL-expressing VRP (1 × 10 6 IU), VRP expressing gHsol / gL (1 × 10 6 Bilateral intramuscular vaccinations (50 μL / leg) were given with either vA160 or vA322 replicons as described above, formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; made using method (A) discussed above, but with a 150 μg RNA batch size).

[0200] The vA160 liposomes had a Zav diameter of 168 nm, a pdI of 0.144, and 87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, a pdI of 0.131, and 90% encapsulation.

[0201] The replicon was capable of expressing two proteins from a single vector.

[0202] On day 63 (3wp3), sera were collected for immunological analysis. CMV neutralization titers (the reciprocal of the serum dilution producing a 50% reduction in the number of positive viral foci / well compared to controls) were as follows:

[0203] [Table 19] RNA expressing either the full-length or soluble forms of the CMV gH / gL complex thus induced high titers of neutralizing antibodies when assayed in epithelial cells. The average titers induced by the liposome-encapsulated RNA were at least as high as those for the corresponding VRP.

[0204] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. The present invention also relates to the following aspects: [1] A liposome having a lipid bilayer encapsulating an aqueous core, where: (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6; and (ii) the aqueous core comprises RNA encoding an immunogen. [2] The liposome according to the above [1], wherein the lipid having a pKa in the range of 5.0 to 7.6 has a tertiary amine. [3] The liposome according to any one of the above [1] to [2], wherein the pKa in the range of 5.0 to 7.6 is between 5.7 and 5.9. [4] The liposome according to [1] above, wherein the lipid having a pKa in the range of 5.0 to 7.6 has the formula given herein for RV01, RV02, RV03, RV04, RV05, RV06, RV07, RV08, RV09, RV11, RV12, RV16 or RV17. [5] The liposome according to any one of the above [1] to [4], which has a diameter in the range of 20 to 220 nm. [6] The liposome according to any one of the above [1] to [5], wherein the RNA molecule encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the RNA molecule, and (ii) an immunogen. [7] The liposome described in [5] above, wherein the RNA molecule has two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen. [8] The liposome according to any one of the above [1] to [7], wherein the RNA molecule has a length of 9,000 to 12,000 nucleotides. [9] The liposome according to any one of the above [1] to [8], wherein the immunogen is capable of inducing an immune response in vivo against a bacterium, a virus, a fungus or a parasite.

[10] The liposome according to any one of the above [1] to [9], wherein the immunogen is capable of inducing an immune response against respiratory syncytial virus glycoprotein F in vivo.

[11] A pharmaceutical composition comprising the liposome according to any one of [1] to

[10] above.

[12] A method for eliciting a protective immune response in a vertebrate, the method comprising the step of administering to the vertebrate an effective amount of the liposome described in [1] to

[10] above, or the pharmaceutical composition described in

[11] above.

[13] A process for preparing RNA-containing liposomes, the process comprising the steps of: (a) mixing RNA with a lipid at a pH below the pKa of the lipid but above 4.5; and then (b) raising the pH above the pKa of the lipid during liposome formation.

[14] The process according to

[13] above, wherein the RNA used in step (a) is in an aqueous solution to be mixed with the organic solution of lipids to give a mixture which is then diluted to form liposomes; and the pH is increased in step (b) after liposome formation.

[0205]

number

[0206]

number

Claims

1. A composition comprising a liposome and an RNA molecule encoding an immunogen, the immunogen comprises a respiratory syncytial virus (RSV) immunogen, a coronavirus spike polypeptide immunogen, or an influenza A virus immunogen; the RNA molecule encoding the immunogen comprises a polyA tail; the liposome has a lipid bilayer that encapsulates at least half of the RNA molecule encoding the immunogen within an aqueous core; the lipid bilayer comprises a lipid comprising a tertiary amine; the lipid containing a tertiary amine has a pKa in the range of 5.8 to 7.0; The pKa of the lipid containing a tertiary amine is in the range of 5.8 to 7.

0. 1) mixing 400 μL of the lipid at 2 mM in 100% ethanol by volume with 800 μL of the fluorescent probe toluene nitrosulfonic acid (TNS) at 0.3 mM in 90% ethanol and 10% methanol by volume, thereby obtaining a lipid / TNS mixture; 2) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a first buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid / buffer mixture, and dispensing 100 μL of the first lipid / buffer mixture into a first well of a 96-well plate with a clear bottom; 3) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a second buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 5.27, thereby obtaining a second lipid / buffer mixture, and dispensing 100 μL of the second lipid / buffer mixture into a second well of the 96-well plate; 4) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a third buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid / buffer mixture, and dispensing 100 μL of the third lipid / buffer mixture into a third well of the 96-well plate; 5) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fourth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid / buffer mixture, and dispensing 100 μL of the fourth lipid / buffer mixture into a fourth well of the 96-well plate; 6) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fifth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid / buffer mixture, and dispensing 100 μL of the fifth lipid / buffer mixture into a fifth well of the 96-well plate; 7) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a sixth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid / buffer mixture, and dispensing 100 μL of the sixth lipid / buffer mixture into a sixth well of the 96-well plate; 8) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a seventh buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid / buffer mixture, and dispensing 100 μL of the seventh lipid / buffer mixture into a seventh well of the 96-well plate; 9) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of an eighth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid / buffer mixture, and dispensing 100 μL of the eighth lipid / buffer mixture into an eighth well of the 96-well plate; 10) measuring absolute fluorescence at a wavelength of 431 nm for each of wells 1 through 8 of the 96-well plate and for each empty well, using an excitation wavelength of 322 nm and a cutoff of less than 420 nm; 11) subtracting the absolute fluorescence of the blank wells from the absolute fluorescence of each of the first through eighth wells, thereby obtaining a blank subtracted fluorescence for each of the first through eighth lipid / buffer mixtures; 12) normalizing each of the blank-removed fluorescence of the first through eighth lipid / buffer mixtures to the blank-removed fluorescence of the first lipid / buffer mixture, thereby obtaining a relative fluorescence for each of the first through eighth lipid / buffer mixtures, where the relative fluorescence of the first lipid / buffer mixture is set to 1; 13) plotting the relative fluorescence of the first through eighth lipid / buffer mixtures versus the pH of the first through eighth lipid / buffer mixtures; 14) Finding the pH corresponding to a relative fluorescence of 0.5 from the graph obtained in 13), and determining the pH corresponding to the relative fluorescence of 0.5 as the pKa of the lipid. Determined by, composition.

2. A composition comprising a liposome and a self-replicating RNA molecule encoding an immunogen, the immunogen comprises a respiratory syncytial virus (RSV) immunogen, a coronavirus spike polypeptide immunogen, or an influenza A virus immunogen; the self-replicating RNA molecule encoding the immunogen comprises a polyA tail; the liposome has a lipid bilayer that encapsulates at least half of a self-replicating RNA molecule encoding the immunogen within an aqueous core; the lipid bilayer comprises a lipid comprising a tertiary amine; the lipid containing a tertiary amine has a pKa in the range of 5.0 to 7.6; The pKa of the lipid containing a tertiary amine is in the range of 5.0 to 7.6, 1) mixing 400 μL of the lipid at 2 mM in 100% ethanol by volume with 800 μL of the fluorescent probe toluene nitrosulfonic acid (TNS) at 0.3 mM in 90% ethanol and 10% methanol by volume, thereby obtaining a lipid / TNS mixture; 2) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a first buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid / buffer mixture, and dispensing 100 μL of the first lipid / buffer mixture into a first well of a 96-well plate with a clear bottom; 3) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a second buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 5.27, thereby obtaining a second lipid / buffer mixture, and dispensing 100 μL of the second lipid / buffer mixture into a second well of the 96-well plate; 4) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a third buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid / buffer mixture, and dispensing 100 μL of the third lipid / buffer mixture into a third well of the 96-well plate; 5) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fourth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid / buffer mixture, and dispensing 100 μL of the fourth lipid / buffer mixture into a fourth well of the 96-well plate; 6) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fifth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid / buffer mixture, and dispensing 100 μL of the fifth lipid / buffer mixture into a fifth well of the 96-well plate; 7) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a sixth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid / buffer mixture, and dispensing 100 μL of the sixth lipid / buffer mixture into a sixth well of the 96-well plate; 8) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a seventh buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid / buffer mixture, and dispensing 100 μL of the seventh lipid / buffer mixture into a seventh well of the 96-well plate; 9) mixing 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of an eighth buffer solution, which is a sodium salt buffer solution containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate and 150 mM sodium chloride and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid / buffer mixture, and dispensing 100 μL of the eighth lipid / buffer mixture into an eighth well of the 96-well plate; 10) measuring absolute fluorescence at a wavelength of 431 nm for each of wells 1 through 8 of the 96-well plate and for each empty well, using an excitation wavelength of 322 nm and a cutoff of less than 420 nm; 11) subtracting the absolute fluorescence of the blank wells from the absolute fluorescence of each of the first through eighth wells, thereby obtaining a blank subtracted fluorescence for each of the first through eighth lipid / buffer mixtures; 12) normalizing each of the blank-removed fluorescence of the first through eighth lipid / buffer mixtures to the blank-removed fluorescence of the first lipid / buffer mixture, thereby obtaining a relative fluorescence for each of the first through eighth lipid / buffer mixtures, where the relative fluorescence of the first lipid / buffer mixture is set to 1; 13) plotting the relative fluorescence of the first through eighth lipid / buffer mixtures versus the pH of the first through eighth lipid / buffer mixtures; 14) Finding the pH corresponding to a relative fluorescence of 0.5 from the graph obtained in 13), and determining the pH corresponding to the relative fluorescence of 0.5 as the pKa of the lipid. Determined by, composition.

3. The composition described in claim 2, wherein the lipid containing a tertiary amine has a pKa in the range of 5.8 to 7.

0.

4. 3. The composition of claim 1 or 2, wherein the tertiary amine-containing lipid has a pKa in the range of 6.07 to 7.

0.

5. The composition according to any one of claims 1 to 4, wherein the liposomes have a diameter in the range of 20 to 220 nm.

6. The composition of claim 2, wherein the self-replicating RNA molecule encoding the immunogen has two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen.

7. The composition of any one of claims 1 to 6, wherein the lipid bilayer further comprises a polyethylene glycol-conjugated (PEG-conjugated) lipid, and / or cholesterol.

8. The composition of any one of claims 1 to 7, wherein the PEG-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].

9. The composition according to any one of claims 1 to 8, wherein the lipid bilayer contains cholesterol in a proportion of 35 mol% to 50 mol% of the total lipids forming the lipid bilayer.

10. The composition of any one of claims 1 to 9, wherein the immunogen comprises two or more immunogens.

11. The lipid bilayer comprises a lipid containing a tertiary amine in a ratio of 40 mol% to 60 mol% of the total lipid forming the lipid bilayer. The composition according to any one of claims 1 to 10.

12. The composition of any one of claims 1 to 11, wherein the lipid bilayer further comprises 1,2-diastearoyl-sn-glycero-3-phosphocholine.

13. The composition of any one of claims 1 to 12, wherein the lipid bilayer encapsulates between 71% and 97.6% of the RNA molecule encoding the immunogen within an aqueous core.

14. The composition of any one of claims 1 to 12, wherein the lipid bilayer encapsulates between 85.3% and 97.6% of the RNA molecule encoding the immunogen within an aqueous core.

15. A pharmaceutical composition comprising the composition according to any one of claims 1 to 14.

16. 16. The pharmaceutical composition of claim 15 for eliciting a protective immune response in a vertebrate.

17. 17. The pharmaceutical composition according to claim 15 or 16, for inducing a cell-mediated immune response against said immunogen and / or an antibody response against said immunogen in vivo.

18. 3. A process for preparing a composition according to claim 1 or 2, comprising the steps of: (i) mixing an RNA molecule encoding the immunogen with a lipid at a pH below the pKa of the lipid but above 4.5; and then (ii) raising the pH above the pKa of the lipid during liposome formation.

19. 20. The process of claim 18, wherein the RNA molecule encoding the immunogen used in step (i) is in an aqueous solution to mix with the organic solution of lipids to result in a mixture which is then diluted to form liposomes; and the pH is increased in step (ii) after liposome formation.

20. Use of a composition according to any one of claims 1 to 14 for the manufacture of a medicament for raising a protective immune response in a vertebrate.

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